Polyamine Oxidase Isoenzymes In Maternal Milk

Polyamine oxidase (PAO) was purified from lactating mother's milk using dialysis, anion exchange chromatography (DEAE-cellulose), gel filtration and SDS- polyacrylamide electrophoresis. Three PAO isoenzymes Ia, Ib and IIa were obtained from normal maternal milk with specific activity of 529.6, 1008.0 and 520.7 unit/ mg protein respectively and with purification fold of 229, 436 and 225 respectively, compared to crude enzyme.
The kinetic parameters of polyamine (spermidine) oxidation by PAO were studied. A linear relationship were obtained between PAO isoenzymes activity and protein concentration up to 60 sec. The optimal pH for the isoenzymes Ia, Ib and IIa were 8.6, 7.2, and 7.2 respectively. The Michaelis Menten constant (km) were and M spermidine respectively.
Phenyl hydrazine was found to inhibit completely the activity of IIa, but showed different inhibitory effects on Ia and Ib activity. Iodoacetamide showed different inhibitory effects on the Ia and IIa activity but showed no inhibitory effect on Ib activity.
It was found that the approximate molecular weight of the isoenzymes Ia, Ib and IIa were 60255, 56234 and 46773 dalton respectively using SDS - polyacrylamide gel electrophoresis.